The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doyle, H. A.
Right arrow Articles by Murphy, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doyle, H. A.
Right arrow Articles by Murphy, J. W.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 1999, 162: 4824-4833.
Copyright © 1999 by The American Association of Immunologists

Role of the C-C Chemokine, TCA3, in the Protective Anticryptococcal Cell-Mediated Immune Response1

Hester A. Doyle2 and Juneann W. Murphy3

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activated T lymphocytes play a crucial role in orchestrating cellular infiltration during a cell-mediated immune (CMI) reaction. TCA3, a C-C chemokine, is produced by Ag-activated T cells and is chemotactic for neutrophils and macrophages, two cell types in a murine CMI reaction. Using a gelatin sponge model for delayed-type hypersensitivity (DTH), we show that TCA3 is a component of the expression phase of an anticryptococcal CMI response in mice. TCA3 mRNA levels are augmented in anticryptococcal DTH reactions at the same time peak influxes of neutrophils and lymphocytes are observed. Neutralization of TCA3 in immunized mice results in reduced numbers of neutrophils and lymphocytes at DTH reaction sites. However, when rTCA3 is injected into sponges in naive mice, only neutrophils are attracted into the sponges, indicating TCA3 is chemotactic for neutrophils, but not lymphocytes. We show that TCA3 is indirectly attracting lymphocytes into DTH-reactive sponges by affecting at least one other chemokine that is chemotactic for lymphocytes. Of the two lymphocyte-attracting chemokines assessed, monocyte-chemotactic protein-1 and macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), only MIP-1{alpha} was reduced when TCA3 was neutralized, indicating that TCA3 affects the levels of MIP-1{alpha}, which attracts lymphocytes into the sponges. TCA3 also plays a role in protection against Cryptococcus neoformans in the lungs and brains of infected mice, as evidenced by the fact that neutralization of TCA3 results in increased C. neoformans CFU in those two organs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C;-5q;44qryptococcusneoformans is a fungal pathogen that causes significant morbidity and mortality in immunocompromised individuals (1, 2). The organism is acquired via inhalation, and in individuals with defective or absent T cell immunity, the organism disseminates to the central nervous system, where it causes life-threatening meningoencephalitis. Currently, there are no antifungal drugs that completely eradicate the organism from the body; thus, cryptococcosis patients must remain on maintenance antifungal therapy for the remainder of their lives (2). Knowledge of the components that are required for a protective host response against C. neoformans would be beneficial to the development of immunomodulatory or immunoreplacement therapies to combat the disease.

Host protection against C. neoformans is highly dependent on the development of a cell-mediated immune (CMI)4 response. Activated T lymphocytes, especially CD4+ T cells, are important components of an anticryptococcal CMI reaction, and they are necessary for the clearance of C. neoformans during infection (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Activated T cells mediate the migration and activation of effector cells such as neutrophils and macrophages, which in an activated state, are more aggressive killers of cryptococci (13, 14). Indeed, clearance of the organism correlates with leukocyte influx during pulmonary cryptococcosis in mice (7, 15, 16, 17). Furthermore, without the influx of these leukocytes into the infected tissue, the organism cannot be eradicated (7).

The means by which activated T lymphocytes influence migration of other leukocytes into the site of a CMI reaction have not been fully established. It is known that activated T cells produce small chemotactic cytokines, called chemokines, that attract leukocytes into tissues (18, 19, 20, 21). One member of the C-C chemokine family that is made by Ag-activated T cells is TCA3 (22). This chemokine has been shown through in vitro and in vivo studies in mice to be chemotactic for neutrophils and macrophages, but not for lymphocytes (23). In addition, TCA3 mediates integrin adhesiveness and activates neutrophils and macrophages (24), properties that would be expected to enhance the clearance of cryptococci. Consequently, we hypothesized that TCA3 would be a component of the anticryptococcal CMI reaction and have a role in protection against C. neoformans.

To gain an understanding of the host components in a protective CMI response, our laboratory has been utilizing a gelatin sponge model of an anticryptococcal delayed-type hypersensitivity (DTH) reaction (25, 26). This model has proved to be a highly reproducible model for studying the efferent or expression phase of a CMI response (27). Unlike the infection model, the CMI reaction occurring in the DTH-reactive sponge is not complicated by other host or organism interactions, so more reliable interpretations can be made concerning mechanisms of the CMI response (27). The reaction that occurs in an Ag-injected gelatin sponge implanted in an immune mouse is similar to the reaction that occurs in an immune animal’s tissue at the site of organism deposition. Histologically, the reaction in the gelatin sponge exhibits the characteristic perivascular cuffing and delayed mononuclear cell infiltrate of a classic DTH reaction (28). Over the 36-h period after Ag injection into the sponges implanted in immune mice, there are two waves of leukocytes that enter the sponges (26). The first wave consists of neutrophils and lymphocytes, with peak numbers appearing 24 h after cryptococcal Ag (CneF) injection (26). A second wave of leukocytes, which includes neutrophils, lymphocytes, and macrophages, starts at 30 h after CneF injection and increases to the endpoint of the study (36 h) (26). The sponge can be easily removed from the animals, and the influxing leukocytes, along with the cytokines and chemokines produced at the reaction site, can be readily obtained for assessment. This makes the gelatin sponge model an excellent one for evaluating the host components involved in leukocyte influx into the site of an anticryptococcal CMI response.

In the current study, we used the anticryptococcal DTH-reactive sponge model to: 1) define the kinetics of TCA3 mRNA production during a DTH reaction, 2) relate the TCA3 mRNA levels to neutrophil, lymphocyte, and macrophage influx into the DTH reaction site, 3) assess the effects of TCA3 on other chemokines known to be present during anticryptococcal CMI responses, and 4) determine the effect of TCA3 in protection against C. neoformans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female inbred CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 7–10 wk of age for these studies.

Maintenance of endotoxin-free conditions

To eliminate the possibility of endotoxin influencing the data, experiments were performed under conditions that minimized endotoxin. These included the use of endotoxin-free plasticware and heating all glassware for 3 h at 180°C. All reagents used were negative for endotoxin in the Limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, MD), which has a minimal detectable level of 8 pg endotoxin/ml.

Ag preparation and analysis

CneF used for immunization and injection of footpads and sponges was prepared from C. neoformans isolate 184A, as previously described (25). Briefly, a defined growth medium (pH 5) was inoculated with 109 yeast cells/L of medium, and the culture was incubated for 5 days at 30°C. The supernatant from this culture was collected with a Millipore OM-141 Pellicon Tangential Flow System and a 0.45-µm pore size cassette (Millipore, Bedford, MA). The supernatant was washed extensively with sterile endotoxin-free physiologic saline solution (SPSS) and was concentrated 10-fold with the Pellicon System fitted with a 30,000 m.w. cutoff cassette. The concentrated retentate designated CneF was filter sterilized and stored at -20°C until used. The CneF preparation used in these studies had a protein concentration of 0.268 mg/ml, as determined by the bicinchoninic acid assay (Pierce, Rockford, IL), and a carbohydrate concentration of 5.8 mg/ml, as determined by the phenol-sulfuric acid assay (29).

Induction and elicitation of the anticryptococcal DTH response

Induction of the anticryptococcal CMI response was performed by immunizing CBA/J mice with CneF emulsified in CFA (26). Briefly, 0.1 ml of a 1:1 emulsion of CneF in CFA was injected s.c. at each of two sites at the base of the tail. At 6 days after immunization, the left hind footpad was injected with 30 µl of SPSS and the right footpad was injected with 30 µl of CneF. The footpads were measured before injection and 24 h after injection of SPSS or CneF. The increase in footpad swelling was calculated by subtracting the difference in swelling in the 0- and 24-h measurements of the SPSS-injected footpads from the difference in swelling between the 0- and 24-h measurements of the CneF-injected footpads. The amount of swelling is directly proportional to the level of an anticryptococcal CMI response (11, 30). Negative control animals were mice injected with SPSS-CFA in place of CneF-CFA and were otherwise treated the same as the immunized mice.

Sponge implantation, injection with Ag, retrieval, and disaggregation

Gelatin sponges (Gelfoam sterile absorbable gelatin sponge; Upjohn, Kalamazoo, MI) were surgically implanted under aseptic conditions (25). Briefly, sponges were cut into 17 x 18 x 10-mm blocks before rehydrating with sterile HBSS containing 100 U penicillin/ml and 100 mg streptomycin/ml. Two days after immunization with CneF-CFA, mice were anesthetized and sponges were implanted s.c. through an incision on the animal’s shaved backs. On day 4 after implantation, one sponge was injected with 0.1 ml CneF, and the other with 0.1 ml SPSS. For controls, mice were injected with SPSS-CFA 2 days before sponge implantation. Otherwise, control mice were treated in the same way as immunized mice. The sponges were removed at the designated times.

Mice were euthanized before surgical removal of sponges. Fluid was expressed from the sponges and stored at -70°C until used for chemokine/cytokine assays. Sponges were put into Stomacher bags (Tekmar, Cincinnati, OH) with enzyme mixture (400 U collagenase/ml; Sigma, St. Louis, MO), then homogenized with three 10-s pulses on a Stomacher (Stomacher 80 Lab Blender; Tekmar) at 15-min intervals (31). During the 15-min intervals, the sponge homogenates were incubated at 37°C. Following the dissagregation step, the sponge homogenates were filtered through 390-µm nylon screens, followed by passage through 140-µm nylon screens, and washed with HBSS. The erythrocytes in the sponge homogenates were lysed by treatment with Tris-NH4Cl (17 mM Tris, 139.7 mM NH4Cl), and the remaining cells were washed once with HBSS. Viable cell counts were made with the use of a hemacytometer and the trypan blue dye exclusion method.

Differential and flow-cytometric analysis of sponge-infiltrating cells

Cytospins of cell suspensions from disaggregated sponges were stained with Harleco Wright Stain, followed by staining in Diff-Quick solutions I and II (Baxter, Miami, FL). The numbers of neutrophils, lymphocytes, macrophages, and eosinophils in each sponge were counted in a total of 200 cells per sample. To determine the total numbers of the designated cell type per sponge, the percentage of that cell type in each sponge was multiplied by the total number of leukocytes in that respective sponge.

Injection of rTCA3 into sponges

Cell influx into sponges in response to rTCA3 was determined by implanting sponges into naive and immunized mice. Because the mice in which we have assessed TCA3 activity have been mice immunized with CneF-CFA, we used a similar immune group in this experiment to assess the chemotactic characteristics of rTCA3. A group of naive mice was also included to determine whether leukocytes in immune mice responded differently to TCA3 than naive leukocytes. Four days after sponge implantation into each group of mice, one sponge was injected with 0.1 ml SPSS, and the other with 0.1 ml of a solution of 210 ng rTCA3/ml (dose injected = 21 ng; PharMingen, San Diego, CA). This dosage was selected based on in vivo studies using several concentrations of rTCA3 (23, 32). In our hands, 21 ng rTCA3 induced the greatest cell influx as compared with SPSS-injected sponges. Six hours after sponge injection was the time selected to assess cell influx in rTCA3-injected sponges based on preliminary experiments demonstrating that cell influx was higher at 6 h than at 3 h after injection of rTCA3. Leukocyte influx at later times was not determined because there are greater chances for the rTCA3 to induce production of other chemokines by the resident macrophages, which are the major cell population in sponges at 4 days after implantation.

In vivo neutralization of TCA3 in the sponge model

Preliminary experiments were done to determine the amount of anti-TCA3 Ab to use for neutralization. The package insert for the Ab indicated that the 100 ng/ml of the Ab neutralized >90% of 100 ng/ml of mouse rTCA3 (PharMingen), so we treated immunized mice with 100, 200, or 500 ng of anti-TCA3 and determined how those doses affected leukocyte influx into the CneF-injected sponges. We found that neither the 200 ng nor 500 ng dose of anti-TCA3 Ab caused a further reduction in leukocyte influx beyond that observed with the 100 ng of anti-TCA3 Ab. We interpreted these data to indicate that 100 ng of anti-TCA3 Ab was neutralizing all of the TCA3 that could be neutralized by this means. Consequently, 100 ng of anti-TCA3 was used in subsequent experiments.

For assessing the effects of neutralizing TCA3, mice were immunized and sponges were implanted as described above. On day 6 after immunization, mice were injected i.p. with 100 ng of neutralizing hamster anti-mouse TCA3 Ab or hamster IgG as a control (PharMingen) in 0.2 ml SPSS. Immediately after Ab injection, one sponge in each mouse was injected with 0.1 ml SPSS, and the other with 0.1 ml CneF. Sponges were removed at the designated times after sponge injection. Fluid was expressed from each sponge and stored at -70°C until used in chemokine assays. Leukocytes from each sponge were collected for differential or flow-cytometric analysis.

MCP-1 and MIP-1{alpha} ELISA

MCP-1 present in fluid expressed from each sponge was quantified with a sandwich ELISA according to manufacturer’s instructions (R&D Systems, Minneapolis, MN). The amount of MIP-1{alpha} in the samples was determined with a sandwich ELISA, as previously described (33).

RNase protection assay

Sponges were removed from immunized mice at designated times after injection of either CneF or saline into the sponges. Total RNA was isolated from the sponge cells using TriReagent (Molecular Research Center, Cincinnati, OH) and frozen at -70°C until assayed. The RNase protection assay was performed according to manufacturer’s instructions (RiboQuant Multi Probe RNase Protection Assay; PharMingen). Briefly, 10 µg of total RNA was hybridized with [32P]UTP-labeled riboprobes for 12–16 h at 56°C. Unhybridized RNA was digested with RNase A and T1. The RNases were then digested by proteinase K treatment. Protected RNA was isolated by phenol/chloroform extraction and sodium acetate/ethanol precipitation. Samples were electrophoresed on a 6% acrylamide/7 M urea gel. Bands were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed using ImageQuant software. Sponge RNA was normalized to L32 (ribosomal subunit protein) RNA.

FITC conjugation of Abs

FITC (Calbiochem, La Jolla, CA) was dissolved at 10 mg/ml in DMSO. To prepare for FITC conjugation, anti-MIP-1{alpha} Ab and goat IgG (R&D Systems) were dialyzed overnight in carbonate buffer (0.160 M Na2CO3, 0.333 M NaHCO3), then the Ab concentrations were adjusted to 1 mg/ml before the addition of 100 µg FITC per mg Ab. The reaction mix was incubated for 45 min at 37°C. The FITC-conjugated Ab was separated from unbound FITC on a PD10 Sepharose column (Pharmacia, Piscataway, NJ) using 0.02 M PBS, pH 7.2, as an eluant.

Intracellular chemokine and surface staining

To determine whether the neutrophils influxing into the sponges were producing MIP-1{alpha}, sponge cells were stained for the neutrophil marker Ly-6G and intracellular MIP-1{alpha}. Mice were immunized and sponges were implanted as described above. Six days after immunization, mice were treated i.p. with 100 ng of anti-TCA3 Ab or hamster IgG as a control. Immediately following Ab injection, one sponge was injected with 0.1 ml CneF, and the other with 0.1 ml SPSS. Sponges were removed 20 h after sponge injection, and the cells were collected as described above, except that all solutions contained 10 µg/ml Brefeldin A (Sigma). Cells from each sponge group were pooled, counted, and resuspended in RPMI + 5% FCS +10 µg/ml Brefeldin A at a concentration of 1 x 107 cells/ml. One hundred microliters of the cell suspension (106 cells) were plated into wells of U-bottom 96-well microtiter plates. The plates were incubated at 37°C for 4 h, then were centrifuged before removing the supernatants. Fc receptors on cells were blocked with 0.1 ml of HB 197 (anti-CD32/CD16) supernatant (American Type Culture Collection, Manassas, VA) + 10 µg/ml Brefeldin A for 30 min on ice. Cells were centrifuged, and the supernatant was removed. Cells were surface stained for 30 min on ice with either 1 µg of phycoerythrin anti-Ly-6G (clone RB6-8C5) (neutrophils) (PharMingen) or phycoerythrin rat IgG2b as an isotype control (Caltag Laboratories, San Francisco, CA). The labeled cells were washed twice with wash buffer (0.02 M PBS (pH 7.2), 1% FCS, and 0.1% NaN3) + 10 µg/ml Brefeldin A before being fixed with 2% paraformaldehyde in wash buffer + Brefeldin A for 20 min at room temperature. Cells were washed once with wash buffer and incubated 10 min at room temperature with wash buffer + 0.1% saponin to permeabilize the cells. The permeabilized cells were then incubated with 10 µg/ml FITC-labeled anti-MIP-1{alpha} Ab or FITC goat IgG as a control for 30 min at room temperature. To control for surface staining, cells from each sponge group were treated with wash buffer and no saponin before being subjected to FITC-labeled anti-MIP-1{alpha} in wash buffer alone with no saponin. The permeabilized labeled cells were washed twice in wash buffer + 0.1% saponin, and the nonpermeabilized labeled cells were washed in buffer alone (surface control). A final wash in wash buffer alone was performed on all groups of cells. The cells were filtered through 30 µm nylon mesh and analyzed on a FACStarPlus flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The number of MIP-1{alpha}-positive staining cells was determined by [(total number of cells from the sponges) x (% positive staining cells - % positive staining cells with the isotope control)].

In vivo neutralization of TCA3 in an infection model with C. neoformans

Mice were immunized with CneF-CFA, as previously described. Six days later, immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (control) (100 ng) immediately before i.v. injection of 105 CFU of C. neoformans 184A. Dilutions of the C. neoformans inoculum were plated on modified Sabouraud’s agar to confirm the infectious dose. The mice were injected again 2 days after infection with the respective Ab. Seven days after infection, lungs, spleens, livers, and brains of the mice were removed, placed individually in sterile Stomacher bags, and homogenized in SPSS (Stomacher 80 Lab Blender; Tekmar). Serial 10-fold dilutions of each organ were made and plated in duplicate on modified Sabouraud’s agar. CFU were counted 2 to 3 days later.

Statistical analysis

Mean, SEM, and unpaired Student’s t test were used to analyze the data. Results were considered significant if the p value was <=0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of TCA3 expression

Mice were immunized with CneF-CFA or injected with SPSS-CFA. The mice immunized with CneF-CFA had activated T cells that recognized cryptococcal Ag because by day 7 after immunization, the mice had a mean increase in footpad thickness of 15.3 ± 0.67 x 10-3 inches in response to CneF (DTH reactivity), whereas the control mice (SPSS-CFA injected) had a mean increase in footpad thickness of 0.4 ± 0.27 x 10-3 inches. To determine the kinetics of TCA3 expression, sponges were implanted into additional mice 2 days after CneF-CFA or SPSS-CFA treatment. Four days after implantation (6 days after CneF-CFA or SPSS-CFA treatment), the sponges were injected with CneF or SPSS, and the levels of TCA3 mRNA were measured in the total RNA extracted from sponge cells at various times after injecting the sponges with CneF or SPSS. Beginning 6 h after sponge injection, TCA3 mRNA expression was significantly increased in the CneF-injected sponges in CneF-CFA-immunized mice (DTH-reactive sponges) compared with SPSS-injected sponges from immunized mice or with SPSS- or CneF-injected sponges in control mice (Fig. 1Go). Elevated levels of TCA3 mRNA in the DTH-reactive sponges when compared with controls were observed at each time point assessed through the 36-h experimental period (Fig. 1Go).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. TCA3 mRNA expression in sponges after injection with CneF or SPSS. Total RNA was extracted from CneF- and SPSS-injected sponges removed at the designated times from mice treated 6 days previously with SPSS-CFA (control) or CneF-CFA (immunized). TCA3 mRNA expression relative to L32 RNA was quantified with an RNase protection assay and a PhosphorImager. Each point represents the mean ± SEM of four mice. p values are equal to or less than the number shown in parentheses when CneF-injected sponges in immunized mice are compared with CneF- or SPSS-injected sponges from control mice or SPSS-injected sponges from CneF-CFA-immunized mice. The data are representative of two experiments.

 
Effect of TCA3 neutralization on cell influx

To assess the role of TCA3 in the anticryptococcal CMI reaction, we determined the effect of treatment with anti-TCA3 Ab on leukocyte influx into sponges undergoing a DTH reaction to CneF. In immunized mice, sponges were implanted and, immediately before sponge injection with CneF or saline, mice were injected i.p. with 100 ng hamster anti-mouse TCA3 Ab or 100 ng hamster IgG as a control. The sponges were removed 24 h after sponge injection, the time at which leukocyte numbers peak in the DTH-reactive sponge (26). The mean total number of leukocytes was significantly decreased in DTH-reactive sponges from anti-TCA3 Ab-treated mice (3.5 ± 0.07 x 106) as compared with mean numbers of leukocytes from similarly treated sponges in IgG-treated mice (4.2 ± 0.04 x 106, p < 0.001). The reduction in the leukocyte numbers after TCA3 neutralization was due in part to a significant decrease in the numbers of neutrophils as compared with neutrophil numbers in the DTH-reactive sponges from IgG-treated control mice (p < 0.001) (Fig. 2GoA). The diminution of neutrophil influx into the DTH-reactive sponges after TCA3 neutralization was in accord with data published by others showing that TCA3 induces neutrophil influx (24). Surprisingly, compared with lymphocyte numbers in IgG-treated immune control mice, the lymphocyte numbers infiltrating into the DTH-reactive sponges in anti-TCA3-Ab-treated immune mice at 24 h after sponge injection were significantly decreased (Fig. 2GoB) (p < 0.001). At the 24-h time period after CneF injection into the sponges, macrophage numbers were similar in the DTH-reactive sponges in the anti-TCA3 Ab-treated group and in the IgG-treated group (Fig. 2GoC). Eosinophil numbers, although decreased in DTH-reactive sponges from anti-TCA3-treated mice at 24 h, were not significantly decreased as compared with DTH-reactive sponges from IgG-treated mice (Fig. 2GoD).



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of TCA3 neutralization on leukocyte influx into sponges. CneF-CFA-immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (control) just before sponge injection with SPSS or CneF. Sponges were removed at 24 h after sponge injection, leukocytes were collected, and differential analyses were performed on cytospin preparations. The percentage of each leukocyte population in the sponge was multiplied by the total number of leukocytes in the sponge to determine the number of each leukocyte population infiltrating the sponge. The data are combined from two experiments using five mice per group per experiment. Vertical lines represent SEM. *, p values < 0.05 as compared with CneF-injected sponges from IgG-treated immunized mice.

 
Effect of rTCA3 on cell influx into sponges

Considering that in vivo studies by other investigators have shown no lymphocyte influx in response to TCA3 (23), we questioned whether the TCA3 in the DTH-reactive sponges was directly chemotactic for lymphocytes or was indirectly affecting lymphocyte influx. To address this, sponges were implanted in immune and naive mice. Four days after implantation, the sponges were injected with either SPSS or 21 ng of rTCA3, then 6 h later, the numbers of leukocytes in the sponges were counted. As expected, the numbers of neutrophils influxing into the rTCA3-injected sponges in immune mice were significantly higher than the numbers of neutrophils influxing into the SPSS-injected sponges in immune mice (Fig. 3GoA) (p = 0.009). There were no significant increases in the numbers of lymphocytes, macrophages, or eosinophils in rTCA3-injected sponges compared with numbers of equivalent cell populations in SPSS-injected sponges (Fig. 3Go, B, C, and D). The data in Fig. 3Go were similar to the data obtained from a similar experiment done in naive mice. Together the results demonstrate that rTCA3 directly mediates neutrophil migration, but does not significantly influence lymphocyte migration into sponges, irrespective of the immune status of the animal.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of rTCA3 on cell migration into gelatin sponges. Sponges implanted in CneF-CFA-immunized mice were injected with 0.1 ml SPSS (control) or 0.1 ml rTCA3 (210 ng/ml) on day 4 after sponge implantation. At 6 h after sponge injection, the sponges were removed, cells were collected, and differential analyses were performed on cytospin preparations. The percentage of each leukocyte population in the sponge was multiplied by the total number of leukocytes in the sponge to determine the number of each leukocyte population infiltrating the sponge. The data are combined from two experiments using five mice per group per experiment. Vertical lines represent SEM. *, p = 0.009 as compared with SPSS-injected controls.

 
Effect of TCA3 neutralization on chemokine levels in DTH-reactive sponges

Because our data indicated that TCA3 was not directly chemotactic for lymphocytes in the sponges, we hypothesized that TCA3 was indirectly affecting lymphocyte migration through the modulation of other chemokines, such as MCP-1 or MIP-1{alpha}, two chemokines known to attract lymphocytes (34, 35, 36, 37). MCP-1 has been shown to be a component of a DTH reaction in skin (38) and has been found to modulate lymphocyte migration during a pulmonary infection with C. neoformans in mice (39). Therefore, TCA3 regulation of MCP-1 was a good possibility in our model. MIP-1{alpha} has been shown to attract T cells, especially Th1 cells (40). In addition, we have shown previously that MIP-1{alpha} is elevated in anticryptococcal DTH-reactive sponges at 24–30 h after sponge injection as compared with controls, and that MIP-1{alpha} mediates lymphocyte migration into DTH-reactive sponges (33). Consequently, MIP-1{alpha} is another candidate for a lymphocyte-attracting chemokine that may be regulated by TCA3.

To determine whether TCA3 influences MCP-1 in the DTH-reactive sponge, we measured the concentration of MCP-1 in sponges from immune mice treated with anti-TCA3 Ab or IgG. MCP-1 protein levels in the DTH-reactive sponges were significantly elevated as compared with MCP-1 levels in control sponges in both the anti-TCA3 Ab- and IgG-treated mice at 12 and 24 h after sponge injection (Fig. 4Go; p = 0.0045), but there were no differences in the levels of MCP-1 between the two Ab-treated groups at either time period (Fig. 4Go).



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of TCA3 neutralization on MCP-1 production in sponges. CneF-CFA-immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (100 ng) (control) just before sponge injection with SPSS or CneF. At 12 or 24 h after sponge injection, sponges were removed and fluid was collected. MCP-1 was measured in the fluid from each sponge by an ELISA that could detect as little as 2 pg MCP-1/ml. The results are representative of two experiments, with five mice per experiment. Vertical lines represent SEM. *, p values <0.05 as compared with SPSS-injected control sponges.

 
To determine whether TCA3 was modulating MIP-1{alpha}, we assessed MIP-1{alpha} mRNA levels with a RNase protection assay and found that MIP-1{alpha} mRNA expression was significantly decreased in DTH-reactive sponges from anti-TCA3 Ab-treated mice at 18 h after sponge injection compared with levels of MIP-1{alpha} mRNA expression in DTH-reactive sponges in IgG-injected mice (Fig. 5GoA) (p = 0.03). MIP-1{alpha} protein levels were measured at 26 h after sponge injection because we had previously observed a peak in MIP-1{alpha} production in the DTH-reactive sponges at 24–30 h after injection of CneF into sponges in immune mice (33). Although the MIP-1{alpha} protein levels in the DTH-reactive sponges in TCA3-treated mice were reproducibly less than those in the DTH-reactive sponges in IgG-treated mice, the difference was not significant at a confidence level of 95%, but if the confidence level was reduced to 94% there would be a significant difference (Fig. 5GoB; p = 0.06).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of TCA3 neutralization on MIP-1{alpha} mRNA and MIP-1{alpha} production in sponges. CneF-CFA-immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (100 ng) (control) just before sponge injection with SPSS or CneF. A, At 18 h after sponge injection, sponges were removed and total RNA to be used in a RNase protection assay was isolated. Each bar represents the mean of four mice; vertical lines show the SEM. B, At 26 h after sponge injection, a time in which MIP-1{alpha} protein levels are elevated in DTH-reactive sponges, sponges were removed and fluid was collected. MIP-1{alpha} was measured in the fluid from each sponge by an ELISA, which could detect as little as 29 pg MIP-1{alpha}/ml. Each bar is the mean result of two experiments, with five mice per group per experiment. Vertical lines represent SEM. *, Significant difference with a p value <0.03 as compared with results in CneF-injected sponges in IgG-treated immune mice.

 
Effects of TCA3 neutralization on MIP-1{alpha}-producing neutrophils

Considering that neutralization of TCA3 in immune mice significantly diminished levels of MIP-1{alpha} mRNA and reproducibly diminished levels of MIP-1{alpha} protein at DTH reaction sites compared with control levels, we were interested in whether TCA3 neutralization affected MIP-1{alpha} levels in the sponges by reducing the numbers of MIP-1{alpha}-producing neutrophils in the DTH-reactive sponges. Because neutrophils are a cell population that can make MIP-1{alpha} (41, 42, 43) and considering neutrophil influx is modulated in our model by TCA3, we determined the effects of TCA3 neutralization on neutrophil production of MIP-1{alpha}. For these determinations, we isolated sponge cells and incubated them in Brefeldin A to stop secretion of the chemokines and cytokines being made by the cells. The cells were stained for Ly-6G (granulocyte marker), fixed, permeabilized, then stained for MIP-1{alpha}. As expected from our previous TCA3 neutralization experiments (Fig. 2Go), the number of granulocytes (Ly-6G+ cells) infiltrating into the DTH-reactive sponges in anti-TCA3-treated mice was 25% less than the number of granulocytes infiltrating into the DTH control sponges (Fig. 6GoA). The number of Ly-6G+ MIP-1{alpha}+ cells in DTH-reactive sponges in anti-TCA3 Ab-treated mice was 35% less than the number of MIP-1{alpha}+ granulocytes in the control Ab-treated animals (Fig. 6GoB). Thus, neutralization of TCA3 reduced the influx of neutrophils into the DTH-reactive sponges, as we have observed previously, but also reduced by 35% the number of MIP-1{alpha}-producing neutrophils. These data considered along with the data showing TCA3 neutralization significantly reduces mRNA for MIP-1{alpha} and reproducibly reduces MIP-1{alpha} protein in DTH-reactive sponges lead us to conclude that TCA3 influences MIP-1{alpha} production, thereby indirectly affecting lymphocyte influx into the sponges.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of TCA3 neutralization on granulocyte+, MIP-1{alpha}-expressing cells from DTH-reactive sponges. CneF-CFA-immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (100 ng) (control) just before sponge injection with SPSS or CneF. At 24 h after sponge injection, sponges were removed. Cells were isolated, pooled, stained for granulocytes with anti-Ly6G mAb and intracellular MIP-1{alpha}, and analyzed by flow cytometry. A, Total number of granulocyte+ sponge cells. B, Number of granulocyte+ sponge cells that also were MIP-1{alpha}+. These data are representative of two separate experiments, with eight mice per group per experiment. Numbers in parentheses indicate the percentage of decrease in cells from DTH-reactive sponges in mice receiving anti-TCA3 Ab as compared with cells from DTH-reactive sponges from IgG-treated control mice.

 
Effect of TCA3 neutralization on clearance of C. neoformans from infected mice

Clearly, TCA3 was effecting leukocyte influx into the site of an anticryptococcal CMI reaction site, so we then asked whether TCA3 plays a role in protection against an infection with C. neoformans. To address this, CneF-CFA-immunized mice were treated with anti-TCA3 Ab or hamster IgG (control) before infecting the mice i.v. with 105 C. neoformans cells. Seven days after infection, there was a significant increase in the number of cryptococcal CFU in the lungs and brains of immune mice treated with anti-TCA3 Ab as compared with C. neoformans CFU in the lungs and brains of immune mice treated with hamster IgG (Fig. 7Go, lung, p = 0.007 and brain, p = 0.046). These findings indicate that TCA3 functions in the clearance mechanism for C. neoformans from lungs and brains. In contrast, TCA3 had little to no impact on clearance from spleen or liver, as shown by cryptococcal CFU in the spleens and livers of immune mice treated with anti-TCA3 Ab being similar to CFU in the respective organs of immune mice treated with hamster IgG (Fig. 7Go).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of TCA3 neutralization on clearance of C. neoformans from lungs, spleen, liver, and brain of CneF-CFA-immunized mice. CneF-CFA-immunized mice were injected i.p. with neutralizing hamster anti-mouse TCA3 Ab (100 ng) or hamster IgG (100 ng) (control) just before i.v. infection with 105 C. neoformans 184A cells. Organs were removed 7 days after infection, homogenized, and plated on modified Sabouraud’s agar to determine the number of CFU. The data are representative of two experiments, with five mice per group per experiment. *, p values <0.05 as compared with IgG-treated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activated T lymphocytes that mediate a CMI reaction are capable of producing chemokines that augment leukocyte infiltration into the infected tissues (19, 20, 21). Our data show that TCA3 is a chemokine whose message is up-regulated in vivo only when activated T lymphocytes are present and after restimulation with the sensitizing Ag. By neutralizing TCA3 at the time of eliciting an anticryptococcal DTH reaction, we demonstrated for the first time in vivo that TCA3 is a component of a DTH reaction and that it influences leukocyte migration into the DTH reaction site. The leukocyte populations attracted into the DTH-reactive sponges as a result in part to the presence of TCA3 are neutrophils and lymphocytes.

TCA3 in the gelatin sponges is directly chemotactic for neutrophils, as shown by the significant influx of neutrophils within 6 h after injecting rTCA3 into sponges implanted in immune or naive mice. Neutralization of TCA3 confirmed that TCA3 in DTH-reactive sponges was influencing the migration of neutrophils, but the TCA3 neutralization data also showed that lymphocyte numbers were clearly being affected by TCA3. We had anticipated that neutrophils would be directly attracted into the DTH reaction site by TCA3 because other investigators have found through both in vivo studies and in vitro chemotactic experiments that TCA3 directly attracts neutrophils and macrophages, but not lymphocytes (23). The numbers of neutrophils attracted into the sponges by rTCA3 were low in comparison with the numbers attracted into the DTH-reactive sponges, but that was an expected outcome and does not affect the interpretation of our results. Any single stimulus injected into a gelatin sponge does not attract as many leukocytes as is attracted into a DTH-reactive sponge (26, 31, 33). This is due to the fact that in the DTH-reactive sponge there are multiple chemokines (Doyle and Murphy, unpublished data) and Ag (31) that can be contributing to the influx of leukocytes.

Based on results of others (23), one would predict that macrophages should have been attracted by the injection of rTCA3 into the sponges; however, that was not the case. At the time sponges are injected with rTCA3, most of the cells in the sponges are macrophages, so if no macrophages or only a few are attracted into the sponges by rTCA3, the differential assessment procedure used is not sufficiently sensitive to detect the change from the control levels. In fact, it appears that rTCA3 preferentially attracted neutrophils at the expense of macrophages, because the macrophage numbers in the rTCA3-injected sponges actually were less than in the sponges injected with saline. The lack of effects of TCA3 neutralization on macrophage numbers in DTH-reactive sponges is in complete agreement with our findings with rTCA3. If TCA3 is regulating macrophage influx into the DTH-reactive sponges, its effects are minimal and not significant, using the differential counts as the assessment method.

TCA3 has not been reported to induce eosinophil influx. In our hands, rTCA3 did not induce any significant eosinophil influx. Furthermore, we obtained no evidence that TCA3 was affecting migration of eosinophils into the DTH-reactive sponges, so it is fair to conclude that TCA3 is not directly or indirectly affecting eosinophil migration into the anticryptococcal DTH reaction site.

The surprise was that lymphocyte numbers in the DTH-reactive sponges were more affected by TCA3 neutralization than were macrophage numbers. Because rTCA3 does not attract lymphocytes into the sponges, we concluded that TCA3 was indirectly influencing the production of other chemokines, which in turn affected lymphocyte migration into the DTH-reactive sponges. Two chemokines, MCP-1 and MIP-1{alpha}, are good candidate chemokines for being modulated by TCA3 because both have displayed chemotactic activity for lymphocytes in reaction sites of C. neoformans or in DTH responses to cryptococcal Ag (33, 39). Huffnagle and coworkers showed that MCP-1 and MIP-1{alpha} attract lymphocytes into lungs of mice infected with C. neoformans (39). We have shown previously that MIP-1{alpha} is chemotactic for lymphocytes in the same anticryptococcal DTH-reactive sponge model used in this study (33). We reasoned that if TCA3 influenced MCP-1 and/or MIP-1{alpha} levels, then TCA3 neutralization would alter the level of the affected chemokine in the DTH-reactive sponges. Consequently, we measured MCP-1 and MIP-1{alpha} levels after treatment of the mice with either anti-TCA3 or control IgG. Although our measurements showed that MCP-1 levels are elevated significantly in DTH-reactive sponges over MCP-1 levels in saline-injected control sponges, neutralization of TCA3 does not affect the levels of MCP-1 in the DTH-reactive sponges, indicating that TCA3 was not working through MCP-1 to draw lymphocytes into DTH-reactive sponges. On the other hand, TCA3 does modulate MIP-1{alpha}, as evidenced by the fact that TCA3 neutralization significantly reduced the levels of MIP-1{alpha} mRNA and routinely diminished from control levels the amount of MIP-1{alpha} protein (significant at 94% confidence level) in the DTH-reactive sponges.

Having found that MIP-1{alpha} was modulated by TCA3 at the DTH reaction site, we were interested in whether the reduction in MIP-1{alpha} after anti-TCA3 treatment was at least in part due to reduced numbers of MIP-1{alpha}-producing neutrophils in the sponges. Other investigators have shown that neutrophils produce MIP-1{alpha} (41, 42, 43), so using flow cytometry, as opposed to differential counts, we evaluated the effects of TCA3 neutralization on the influx of neutrophils into DTH-reactive sponges and on the percentage of MIP-1{alpha}-producing neutrophils among the leukocytes that entered the sponges. As would be expected from the data from the other TCA3 neutralization experiments presented in this work, the numbers of neutrophils entering the sponges are reduced in DTH-reactive sponges when TCA3 is neutralized. Moreover, neutralization of TCA3 results in diminished numbers of neutrophils making MIP-1{alpha} in the DTH-reactive sponges. Consequently, we concluded that TCA3 modulates MIP-1{alpha} levels in the sponges by attracting neutrophils that have the potential to produce MIP-1{alpha}. Because lymphocyte migration has been shown to be dependent on the concentration of MIP-1{alpha} (37, 44), any fluctuations in the levels of MIP-1{alpha}, as we observed in the DTH-reactive sponges in which TCA3 was neutralized, would be expected to affect lymphocyte migration into the sponges, and indeed it did.

Our findings on TCA3 modulation of MIP-1{alpha} add to the growing knowledge of chemokine networks during an anticryptococcal DTH response. Huffnagle and colleagues (45) neutralized MCP-1 during a pulmonary infection with C. neoformans in mice and observed a reduction in levels of MIP-1{alpha} in infected lungs. In parallel studies, they found that neutralization of MIP-1{alpha} did not alter MCP-1 production, so they concluded that MCP-1 was regulating MIP-1{alpha} production (45). Our data show that TCA3 affects MIP-1{alpha} production, but not MCP-1 production, suggesting that TCA3 is working either in parallel with or at a point distal to MCP-1, but before MIP-1{alpha}. MCP-1, shown to attract CD4+ T cells into C. neoformans-infected lung (39), may also attract T cells into the DTH-reactive sponge. MIP-1{alpha} is clearly a component of the anticryptococcal CMI response (33). From combined data from several studies, we hypothesize that the following sequence of events occurs at a DTH reaction site. The cryptococcal Ag is chemotactic for neutrophils (31, 46), so it would induce the first wave of neutrophil influx into the sponges (26). Some activated T cells also enter the sponge during the early phase of the response (26), and those activated T cells have the potential to make TCA3 (22). The TCA3 could attract more neutrophils into the sponges. The neutrophils in the sponge are stimulated to produce MIP-1{alpha}, which in turn attracts more lymphocytes into the sponges (33). All considered, it is very likely that the pathway also has MCP-1 working in combination with TCA3 to influence lymphocyte migration into the DTH-reactive sponges, because lymphocyte numbers are neither completely reduced to background numbers in the DTH-reactive sponges from anti-TCA3 Ab-treated mice nor are MIP-1{alpha} levels reduced to background levels. This hypothesis does not exclude the possibility that other chemokines may be working in combination with TCA3, MIP-1{alpha}, and MCP-1 to mediate leukocyte migration into the DTH reaction site. In fact, we have unpublished data showing the presence of mRNAs for four other chemokines in the DTH-reactive sponges (Doyle and Murphy, unpublished data).

Cellular infiltration is a crucial step in the clearance of C. neoformans from infected tissues. Animals that have developed a CMI response to cryptococcal Ags display more pronounced cellular infiltrates at sites of C. neoformans Ag deposition than seen in nonimmune animals (26). Furthermore, the ability of the leukocytes to infiltrate infected tissues in the immune animals correlates with protection (7, 15, 16, 17). Clearance of cryptococci from infected tissues ultimately results from the interaction of chemokines, cytokines, and leukocytes. The fact that TCA3 mediates the migration of neutrophils and lymphocytes, two cell populations known to either directly (13, 47, 48) and/or indirectly (49) kill cryptococci, suggests that TCA3 contributes to protection against C. neoformans. To test this theory, we injected immune mice with anti-TCA3 Ab or hamster IgG as a control before i.v. infection with C. neoformans. We chose the i.v. route of infection to mimic the hematogenous spread of the organism from the lungs to the spleen, liver, and eventually the brain. TCA3 neutralization in immunized mice infected with cryptococci did not affect the fungal burden in the spleen or liver. It should be noted that in immunized mice the protection afforded by the immunization cannot always be detected by a significant reduction in cryptococcal CFU counts in spleen and liver (33, 50). Consequently, if there is not a significant lowering of CFU in a tissue of immunized mice as compared with nonimmune controls, then neutralization of TCA3 would not be expected to have a measurable effect on clearance of the organism from that tissue. Neutralization of TCA3 with anti-TCA3 Ab did, however, result in significantly reduced clearance of C. neoformans from the lungs as compared with clearance from IgG-treated infected mice, indicating that TCA3 is important for clearance in the lungs of immunized mice. TCA3 also plays a role in the reduction of C. neoformans in the brains of immunized mice because we observed increased numbers of cryptococci in the brains of mice treated with anti-TCA3 Ab over the numbers of cryptococcal CFU in the IgG-treated mice. TCA3 could affect reduction of C. neoformans in the brain by 1) indirectly reducing C. neoformans numbers by enhancing clearance in the lungs, thus preventing seeding of the brain, or 2) directly mediating migration of leukocytes that are capable of clearing cryptococci from the brain. The latter explanation is feasible because TCA3 mRNA has been detected in the central nervous system of mice undergoing the CMI response associated with experimental allergic encephalomyelitis (51).

In summary, we have demonstrated in this study that TCA3 is a component of the expression phase of an anticryptococcal CMI response, and that it plays a role in mediating direct and indirect neutrophil migration and indirect lymphocyte migration into the DTH reaction site. Our data suggest that the indirect route by which TCA3 influences lymphocyte migration into a CMI reaction site is at least in part by the regulation of the numbers of MIP-1{alpha}-producing neutrophils at the site of Ag deposition. In addition, TCA3 contributes to protection against C. neoformans in the lungs and brains of infected mice. A better understanding of the interactions of various chemokines during an immune response may prove useful in developing therapies to modulate CMI-mediated diseases to the benefit of the host.


   Acknowledgments
 
We thank Fredda Schafer for assistance with sponge experiments, Melissa Morrison for assistance with infection studies, and Jim Henthorn for assistance with flow-cytometric analyses. Flow-cytometric analyses were performed at the Flow Cytometry and Cell Sorting Core Facility for Molecular Medicine and St. Francis Research Institute, University of Oklahoma Health Sciences Center (Oklahoma City, OK).


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants AI-15716, AI-18895, and T32-AI-07364. Back

2 Current address: Department of Internal Medicine, Section of Rheumatology, Yale University School of Medicine, 333 Cedar Street, LCI 600, P.O. Box 208031, New Haven, CT 06520. Back

3 Address correspondence and reprint requests to Dr. Juneann Murphy, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, BMSB 1053, Oklahoma City, OK 73190. E-mail address: Back

4 Abbreviations used in this paper: CMI, cell-mediated immune; CneF, cryptococcal culture filtrate Ag; DTH, delayed-type hypersensitivity; MCP, monocyte-chemotactic protein; MIP, macrophage-inflammatory protein; SPSS, sterile endotoxin-free physiologic saline solution. Back

Received for publication October 13, 1998. Accepted for publication January 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kwon-Chung, K. J.. 1992. Cryptococcosis. K. J. Kwon-Chung, and J. E. Bennet, eds. Medical Mycology 397. Lea & Febiger, Philadelphia.
  2. Mitchell, T. G., J. R. Perfect. 1995. Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans. Clin. Microbiol. Rev. 8:515.[Abstract]
  3. Cauley, L. K., J. W. Murphy. 1979. Response of congenitally athymic (nude) and phenotypically normal mice to Cryptococcus neoformans infection. Infect. Immun. 23:644.[Abstract/Free Full Text]
  4. Graybill, J. R., D. J. Drutz. 1978. Host defense in cryptococcosis in the nude mouse. Cell. Immunol. 40:263.[Medline]
  5. Nishimura, K., M. Miyaji. 1979. Histopathological studies on experimental cryptococcosis in nude mice. Mycopathologia 68:145.[Medline]
  6. Huffnagle, G. B., J. L. Yates, M. F. Lipscomb. 1991. T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice. Infect. Immun. 59:1423.[Abstract/Free Full Text]
  7. Huffnagle, G. B., M. F. Lipscomb, J. A. Lovchik, K. A. Hoag, N. E. Street. 1994. The role of CD4+ and CD8+ T cells in the protective inflammatory response to a pulmonary cryptococcal infection. J. Leukocyte Biol. 55:35.[Abstract]
  8. Hill, J. O., A. G. Harmsen. 1991. Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted of CD4+ and CD8+ T cells. J. Exp. Med. 173:755.[Abstract/Free Full Text]
  9. Hill, J. O.. 1992. CD4+ T cells cause multinucleated giant cells to form around Cryptococcus neoformans and confine the yeast within the primary site of infection in the respiratory tract. J. Exp. Med. 175:1685.[Abstract/Free Full Text]
  10. Hill, J. O., K. M. Aguirre. 1994. CD4+ T cell-dependent acquired state of immunity that protects the brain against Cryptococcus neoformans. J. Immunol. 152:2344.[Abstract]
  11. Mody, C. H., M. F. Lipscomb, N. E. Street, G. B. Towes. 1990. Depletion of CD4+ (L3T4+) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans. J. Immunol. 144:1472.[Abstract]
  12. Yuan, R. R., A. Casadevall, J. Oh, M. D. Scharff. 1997. T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice. Proc. Natl. Acad. Sci. USA 94:2483.[Abstract/Free Full Text]
  13. Miller, M. F., T. G. Mitchell. 1991. Killing of Cryptococcus neoformans strains by human neutrophils and monocytes. Infect. Immun. 59:24.[Abstract/Free Full Text]
  14. Chaturvedi, V., B. Wong, S. L. Neuman. 1996. Oxidative killing of Cryptococcus neoformans by human neutrophils: evidence that fungal mannitol protects by scavenging reactive oxygen intermediates. J. Immunol. 156:3886.
  15. Huffnagle, G. B., G. B. Toews, M. D. Burdick, M. B. Boyd, K. S. McAllister, R. A. McDonald, S. L. Kunkel, R. M. Strieter. 1996. Afferent phase production of TNF-{alpha} is required for the development of protective T cell immunity to Cryptococcus neoformans. J. Immunol. 157:4529.[Abstract]
  16. Huffnagle, G. B., G. Chen, J. L. Curtis, R. A. McDonald, R. M. Strieter, G. B. Toews. 1995. Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans. J. Immunol. 155:3507.[Abstract]
  17. Hoag, K. A., N. E. Street, G. B. Huffnagle, M. F. Lipscomb. 1995. Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice. Am. J. Respir. Cell Mol. Biol. 13:487.[Abstract]
  18. Rollins, B. J.. 1997. Chemokines. Blood 90:909.[Free Full Text]
  19. Kelner, G. S., J. Kennedy, K. B. Bacon, S. Kleyensteuber, D. A. Largaespada, N. A. Jenkins, N. G. Copeland, J. F. Bazan, K. W. Moore, T. J. Schall, A. Zlotnik. 1994. Lymphotactin: a cytokine that represents a new class of chemokines. Science 266:1395.[Abstract/Free Full Text]
  20. Schall, T. J., J. Jongstra, B. J. Dyer, J. Jorgensen, C. Clayberger, M. M. Davis, A. M. Krensky. 1988. A human T cell-specific molecule is a member of a new gene family. J. Immunol. 141:1018.[Abstract]
  21. Conlon, K., A. Lloyd, U. Chattopadhyay, N. Lukas, S. Kunkel, T. Schall, D. Taub, C. Morimoto, J. Osborne, J. Oppenheim, H. Young, D. Kelvin, J. Ortaldo. 1995. CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1{alpha}, interleukin-8 and RANTES. Eur. J. Immunol. 25:751.[Medline]
  22. Wilson, S. D., P. R. Burd, P. R. Billings, C. A. Martin, M. E. Dorf. 1988. The expression and regulation of a potential lymphokine gene (TCA3) in CD4 and CD8 T cell clones. J. Immunol. 141:1563.[Abstract]
  23. Luo, Y., J. Laning, S. Devi, J. Mak, T. J. Schall, M. E. Dorf. 1994. Biologic activities of the murine ß-chemokine TCA3. J. Immunol. 153:4616.[Abstract]
  24. Devi, S., J. Laning, Y. Luo, M. E. Dorf. 1995. Biologic activities of the ß-chemokine TCA3 on neutrophils and macrophages. J. Immunol. 154:5376.[Abstract]
  25. Buchanan, K. L., J. W. Murphy. 1993. Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. Infect. Immun. 61:2854.[Abstract/Free Full Text]
  26. Buchanan, K. L., J. W. Murphy. 1997. Kinetics of cellular infiltration and cytokine production during the efferent phase of a delayed-type hypersensitivity reaction. Immunology 90:189.[Medline]
  27. Murphy, J. W.. 1998. Protective cell-mediated immunity against Cryptococcus neoformans. Res. Immunol. 149:373.[Medline]
  28. Goust, J., B. Bierer. 1993. Cell-mediated immunity. Immunol. Ser. 58:187.[Medline]
  29. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, R. Smith. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28:350.
  30. Lim, T. S., J. W. Murphy, L. K. Cauley. 1980. Host-etiological agent interactions in intranasally and intraperitoneally induced cryptococcosis in mice. Infect. Immun. 29:633.[Abstract/Free Full Text]
  31. Dong, Z. M., J. W. Murphy. 1995. Intravascular cryptococcal culture filtrate (CneF) and its major component, glucuronoxylomannan, are potent inhibitors of leukocyte accumulation. Infect. Immun. 63:770.[Abstract]
  32. Wilson, S. D., V. K. Duchroo, D. I. Israel, M. E. Dorf. 1990. Expression and characterization of TCA3: a murine inflammatory protein. J. Immunol. 145:2745.[Abstract]
  33. Doyle, H. A., J. W. Murphy. 1997. MIP-1{alpha} contributes to the anticryptococcal delayed-type hypersensitivity reaction and protection against Cryptococcus neoformans. J. Leukocyte Biol. 61:147.[Abstract]
  34. Carr, M. W., S. J. Roth, E. Luther, S. S. Rose, T. A. Springer. 1994. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc. Natl. Acad. Sci. USA 91:3652.[Abstract/Free Full Text]
  35. Loetscher, P., M. Seitz, I. Clark-Lewis, M. Baggiolini, B. Moser. 1994. Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 8:1055.[Abstract]
  36. Taub, D. D., A. R. Lloyd, J. Wang, J. J. Oppenheim, D. J. Kelvin. 1993. The effects of human recombinant MIP-1{alpha}, MIP-1ß, and RANTES on the chemotaxis and adhesion of T cell subsets. I. J. D. Lindley, ed. The Chemokines 139. Plenum Press, New York.
  37. Schall, T. J., K. Bacon, R. D. R. Camp, J. W. Kaspari, D. V. Goeddel. 1993. Human macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) and MIP-1ß chemokines attract distinct populations of lymphocytes. J. Exp. Med. 177:1821.[Abstract/Free Full Text]
  38. Rand, M. L., J. S. Warren, M. K. Mansour, W. Newman, D. J. Ringler. 1996. Inhibition of T cell recruitment and cutaneous delayed-type hypersensitivity-induced inflammation with antibodies to monocyte chemoattractant protein-1. Am. J. Pathol. 148:855.[Abstract]
  39. Huffnagle, G. B., R. M. Strieter, T. J. Standiford, R. A. McDonald, M. D. Burdick, S. L. Kunkel, G. B. Toews. 1995. The role of monocyte chemotactic protein-1 (MCP-1) in the recruitment of monocytes and CD4+ T cells during a pulmonary Cryptococcus neoformans infection. J. Immunol. 155:4790.[Abstract]
  40. Siveke, J. T., A. Hamann. 1998. Cutting edge: T helper 1 and T helper 2 cells respond differentially to chemokines. J. Immunol. 160:550.[Abstract/Free Full Text]
  41. Kasama, T., R. M. Strieter, T. J. Standiford, M. D. Burdick, S. L. Kunkel. 1993. Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1{alpha}. J. Exp. Med. 178:63.[Abstract/Free Full Text]
  42. McColl, S. R., C. J. Roberge, B. Larochelle, J. Gosselin. 1997. EBV induces the production and release of IL-8 and macrophage inflammatory protein-1{alpha} in human neutrophils. J. Immunol. 159:6164.[Abstract]
  43. VanOtteren, G. M., T. J. Standiford, S. L. Kunkel, J. M. Danforth, M. D. Burdick, L. V. Abruzzo, R. M. Strieter. 1994. Expression and regulation of macrophage inflammatory protein-1{alpha} by murine alveolar and peritoneal macrophages. Am. J. Respir. Cell Mol. Biol. 10:8.[Abstract]
  44. Taub, D. D., K. Conlon, A. R. Lloyd, J. J. Oppenheim, D. J. Kelvin. 1993. Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1{alpha} and MIP-1ß. Science 260:355.[Abstract/Free Full Text]
  45. Huffnagle, G. B., R. M. Strieter, L. K. McNeil, R. A. McDonald, M. D. Burdick, S. L. Kunkel, G. B. Toews. 1997. Macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) is required for the efferent phase of pulmonary cell-mediated immunity to a Cryptococcus neoformans infection. J. Immunol. 159:318.[Abstract]
  46. Dong, Z. M., J. W. Murphy. 1993. Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen. Infect. Immun. 61:5067.[Abstract/Free Full Text]
  47. Fung, P. Y. S., J. W. Murphy. 1982. In vitro interactions of immune lymphocytes and Cryptococcus neoformans. Infect. Immun. 36:1128.[Abstract/Free Full Text]
  48. Muth, S. M., J. W. Murphy. 1995. Direct anticryptococcal activity of lymphocytes from Cryptococcus neoformans-immunized mice. Infect. Immun. 63:1637.[Abstract]
  49. Murphy, J. W.. 1996. Cell-mediated immunity. D. H. Howard, and J. D. Miller, eds. The Mycota VI Human and Animal Relationships 67. Springer-Verlag, Berlin.
  50. Murphy, J. W., F. Schafer, A. Casadevall, A. Adesina. 1998. Antigen-induced protective and nonprotective cell-mediated immune components against Cryptococcus neoformans. Infect. Immun. 66:2632.[Abstract/Free Full Text]
  51. Godiska, R., D. Chantry, G. N. Dietsch, P. W. Gray. 1995. Chemokine expression in murine experimental allergic encephalomyelitis. J. Neuroimmunol. 58:167.[Medline]



This article has been cited by other articles:


Home page
 BloodHome page
N. S. Haque, J. T. Fallon, J. J. Pan, M. B. Taubman, and P. C. Harpel
Chemokine receptor-8 (CCR8) mediates human vascular smooth muscle cell chemotaxis and metalloproteinase-2 secretion
Blood, February 15, 2004; 103(4): 1296 - 1304.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. K. Bauman, G. B. Huffnagle, and J. W. Murphy
Effects of Tumor Necrosis Factor Alpha on Dendritic Cell Accumulation in Lymph Nodes Draining the Immunization Site and the Impact on the Anticryptococcal Cell-Mediated Immune Response
Infect. Immun., January 1, 2003; 71(1): 68 - 74.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
T. M. Tumpey, R. Fenton, S. Molesworth-Kenyon, J. E. Oakes, and R. N. Lausch
Role for Macrophage Inflammatory Protein 2 (MIP-2), MIP-1{alpha}, and Interleukin-1{alpha} in the Delayed-Type Hypersensitivity Response to Viral Antigen
J. Virol., July 17, 2002; 76(16): 8050 - 8057.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
K. L. Nichols, S. K. Bauman, F. B. Schafer, and J. W. Murphy
Differences in Components at Delayed-Type Hypersensitivity Reaction Sites in Mice Immunized with Either a Protective or a Nonprotective Immunogen of Cryptococcus neoformans
Infect. Immun., February 1, 2002; 70(2): 591 - 600.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
S. W. Chensue
Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions
Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. K. Bauman, K. L. Nichols, and J. W. Murphy
Dendritic Cells in the Induction of Protective and Nonprotective Anticryptococcal Cell-Mediated Immune Responses
J. Immunol., July 1, 2000; 165(1): 158 - 167.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doyle, H. A.
Right arrow Articles by Murphy, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doyle, H. A.
Right arrow Articles by Murphy, J. W.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS